cGMP-binding, cGMP-specific phosphodiesterase materials and methods

ABSTRACT

The present invention provides novel purified and isolated nucleotide sequences encoding the cGMP-binding, cGMP-specific phosphodiesterase designated cGB-PDE. Also provided by the invention are methods and materials for the recombinant production of cGB-PDE polypeptide products and methods for identifying compounds which modulate the enzymatic activity of cGB-PDE polypeptides.

This application is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 08/068,051 filed May 27, 1993.

Experimental work described herein was supported in part by ResearchGrants GM15731, DK21723, DK40029 and GM41269 and the Medical ScientistTraining Program Grant GM07347 awarded by the National Institutes ofHealth. The United States government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates generally to a cyclic guanosinemonophosphate-binding, cyclic guanosine monophosphate-specificphosphodiesterase designated cGB-PDE and more particularly to novelpurified and isolated polynucleotides encoding cGB-PDE polypeptides, tomethods and materials for recombinant production of cGB-PDEpolypeptides, and to methods for identifying modulators of cGB-PDEactivity.

BACKGROUND

Cyclic nucleotide phosphodiesterases (PDEs) that catalyze the hydrolysisof 3′5′ cyclic nucleotides such as cyclic guanosine monophosphate (cGMP)and cyclic adenosine monophosphate (cAMP) to the correspondingnucleoside 5′ monophosphates constitute a complex family of enzymes. Bymediating the intracellular concentration of the cyclic nucleotides, thePDE isoenzymes function in signal transduction pathways involving cyclicnucleotide second messengers.

A variety of PDEs have been isolated from different tissue sources andmany of the PDEs characterized to date exhibit differences in biologicalproperties including physicochemical properties, substrate specificity,sensitivity to inhibitors, immunological reactivity and mode ofregulation. [See Beavo et al., Cyclic Nucleotide Phosphodiesterases:Structure, Regulation and Drug Action, John Wiley & Sons, Chichester,U.K. (1990)] Comparison of the known amino acid sequences of variousPDEs indicates that most PDEs are chimeric multidomain proteins thathave distinct catalytic and regulatory domains. [See Charbonneau, pp.267-296 in Beavo et al., supra] All mammalian PDEs characterized to dateshare a sequence of approximately 250 amino acid residues in length thatappears to comprise the catalytic site and is located in the carboxylterminal region of the enzyme. PDE domains that interact with allostericor regulatory molecules are thought to be located within theamino-terminal regions of the isoenzymes. Based on their biologicalproperties, the PDEs may be classified into six general families: theCa²⁺/calmodulin-stimulated PDEs (Type I), the cGMP-stimulated PDEs (TypeII), the cGMP-inhibited PDEs (Type II), the cAMP-specfic PDEs (Type IV),the cGMP-specific phosphodiesterase cGB-PDE (Type V) which is thesubject of the present invention and the cGMP-specific photoreceptorPDEs (Type VI).

The cGMP-binding PDEs (Type II, Type V and Type VI PDEs), in addition tohaving a homologous catalytic domain near their carboxyl terminus, havea second conserved sequence which is located closer to their aminoterminus and which may comprise an allosteric cGMP-binding domain. SeeCharbonneau et al., Proc. Natl. Acad. Sci. USA, 87: 288-292 (1990).

The Type II cGMP-stimulated PDEs (cGs-PDEs) are widely distributed indifferent tissue types and are thought to exist as homodimers of 100-105kDa subunits. The cGs-PDEs respond under physiological conditions toelevated cGMP concentrations by increasing the rate of cAMP hydrolysis.The amino acid sequence of a bovine heart cGs-PDE and a partial cDNAsequence of a bovine adrenal cortex cGS-PDE are reported in LeTrong etal., Biochemistry, 29: 10280-10288 (1990) and full length bovine adrenaland human fetal brain cGB-PDE cDNA sequences are described in PatentCooperation Treaty International Publication No. WO 92/18541 publishedon Oct. 29, 1992. The full length bovine adrenal cDNA sequence is alsodescribed in Sonnenburg et al., J. Biol. Chem., 266: 17655-17661 (1991).

The photoreceptor PDEs and the cGB-PDE have been described ascGMP-specific PDEs because they exhibit a 50-fold or greater selectivityfor hydrolyzing cGMP over cAMP.

The photoreceptor PDEs are the rod outer segment PDE (ROS-PDE) and thecone PDE (COS-PDE). The holoenzyme structure of the ROS-PDE consists oftwo large subunits α (88 kDa) and β (84 kDa) which are bothcatalytically active and two smaller γ regulatory subunits (both 11kDa). A soluble form of the ROS-PDE has also been identified whichincludes α, β, and γ subunits and a δ subunit (15 kDa) that appears tobe identical to the COS-PDE 15 kDa subunit. A full-length cDNAcorresponding to the bovine membrane-associated ROS-PDE α subunit isdescribed in Ovchinnikov et al., FEBS Lett., 223: 169-173 (1987) and afull length cDNA corresponding to the bovine rod outer segment PDE βsubunit is described in Lipkin et al., J. Biol. Chem., 265: 12955-12959(1990). Ovchinnikov et al., FEBS Lett., 204: 169-173 (1986) presents afull-length cDNA corresponding to the bovine ROS-PDE γ subunit and theamino acid sequence of the δ subunit. Expression of the ROS-PDE has alsobeen reported in brain in Collins et al., Genomics, 13: 698-704 (1992).The COS-PDE is composed of two identical α′ (94 kDa) subunits and threesmaller subunits of 11 kDa, 13 kDa and 15 kDa. A full-length cDNAcorresponding to the bovine COS-PDE α′ subunit is reported in Li et al.,Proc. Natl. Acad. Sci. USA, 87: 293-297 (1990).

cGB-PDE has been purified to homogeneity from rat [Francis et al.,Methods Enzymol., 159: 722-729 (1988)] and bovine lung tissue [Thomas etal., J. Biol. Chem., 265: 14964-14970 (1990), hereinafter “Thomas I”].The presence of this or similar enzymes has been reported in a varietyof tissues and species including rat and human platelets [Hamet et al.,Adv. Cyclic Nucleotide Protein Phosphorylation Res., 16: 119-136(1984)], rat spleen [Coquil et al., Biochem. Biophys. Res. Commun., 127:226-231 (1985)], guinea pig lung [Davis et al., J. Biol. Chem., 252:4078-4084 (1977)], vascular smooth muscle [Coquil et al., Biochim.Biophys. Acta, 631: 148-165 (1980)], and sea urchin sperm [Francis etal., J. Biol. Chem., 255: 620-626 (1979)]. cGB-PDE may be a homodimercomprised of two 93 kDa subunits. [See Thomas I, supra] cGB-PDE has beenshown to contain a single site not found in other known cGMP-bindingPDEs which is phosphorylated by cGMP-dependent protein kinase (cGK) and,with a lower affinity, by cAMP-dependent protein kinase (cAK). [SeeThomas et al., J. Biol. Chem., 265: 14971-14978 (1990), hereinafter“Thomas II”] The primary amino acid sequence of the phosphorylation siteand of the amino-terminal end of a fragment generated by chymotrypticdigestion of cGB-PDE are described in Thomas II, supra, and Thomas I,supra, respectively. However, the majority of the amino acid sequence ofcGB-PDE has not previously been described.

Various inhibitors of different types of PDEs have been described in theliterature. Two inhibitors that exhibit some specificity for Type V PDEsare zaprinast and dipyridamole. See Francis et al., pp. 117-140 in Beavoet al., supra.

Elucidation of the DNA and amino acid sequences encoding the cGB-PDE andproduction of cGB-PDE polypeptide by recombinant methods would provideinformation and material to allow the identification of novel agentsthat selectively modulate the activity of the cGB-PDEs. The recognitionthat there are distinct types or families of PDE isoenzymes and thatdifferent tissues express different complements of PDEs has led to aninterest in the development of PDE modulators which may have therapeuticindications for disease states that involve signal transduction pathwaysutilizing cyclic nucleotides as second messengers. Various selective andnon-selective inhibitors of PDE activity are discussed in Murray et al.,Biochem. Soc. Trans., 20(2): 460-464 (1992). Development of PDEmodulators without the ability to produce a specific PDE by recombinantDNA techniques is difficult because all PDEs catalyze the same basicreaction, have overlapping substrate specificities and occur only intrace amounts. As a result, purification to homogeneity of many PDEs isa tedious and difficult process.

There thus continues to exist a need in the art for DNA and amino acidsequence information for the cGB-PDE, for methods and materials for therecombinant production of cGB-PDE polypeptides and for methods foridentifying specific modulators of cGB-PDE activity.

SUMMARY OF THE INVENTION

The present invention provides novel purified and isolatedpolynucleotides (e.g., DNA sequences and RNA transcripts, both sense andantisense strands, including splice variants thereof) encoding thecGMP-binding, cGMP-specific PDE designated cGB-PDE. Preferred DNAsequences of the invention include genomic and cDNA sequences as well aswholly or partially chemically synthesized DNA sequences. DNA sequencesencoding cGB-PDE that are set out in SEQ ID NO: 9 or 20 and DNAsequences which hybridize thereto under stringent conditions or DNAsequences which would hybridize thereto but for the redundancy of thegenetic code are contemplated by the invention. Also contemplated by theinvention are biological replicas (i.e., copies of isolated DNAsequences made in vivo or in vitro) of DNA sequences of the invention.Autonomously replicating recombinant constructions such as plasmid andviral DNA vectors incorporating cGB-PDE sequences and especially vectorswherein DNA encoding cGB-PDE is operatively linked to an endogenous orexogenous expression control DNA sequence and a transcriptionalterminator are also provided. Specifically illustrating expressionplasmids of the invention is the plasmid hcgbmet156-2 6n in E. colistrain JM109 which was deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, on May 4,1993 as Accession No. 69296.

According to another aspect of the invention, host cells includingprocaryotic and eucaryotic cells, are stably transformed with DNAsequences of the invention in a manner allowing the desired polypeptidesto be expressed therein. Host cells expressing cGB-PDE products canserve a variety of useful purposes. Such cells constitute a valuablesource of immunogen for the development of antibody substancesspecifically immunoreactive with cGB-PDE. Host cells of the inventionare conspicuously useful in methods for the large scale production ofcGB-PDE polypeptides wherein the cells are grown in a suitable culturemedium and the desired polypeptide products are isolated from the cellsor from the medium in which the cells are grown by, for example,immunoaffinity purification.

cGB-PDE products may be obtained as isolates from natural cell sourcesor may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofmammalian host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the invention.cGB-PDE products of the invention may be full length polypeptides,fragments or variants. Variants may comprise cGB-PDE polypeptide analogswherein one or more of the specified (i.e., naturally encoded) aminoacids is deleted or replaced or wherein one or more nonspecified aminoacids are added:

(1) without loss of one or more of the biological activities orimmunological characteristics specific for cGB-PDE; or (2) with specificdisablement of a particular biological activity of cGB-PDE.

Also comprehended by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies, CDR-grafted antibodies and the like) and otherbinding proteins specific for cGB-PDE. Specific binding proteins can bedeveloped using isolated or recombinant cGB-PDE or cGB-PDE variants orcells expressing such products. Binding proteins are useful, in turn, incompositions for immunization as well as for purifying cGB-PDEpolypeptides and detection or quantification of cGB-PDE polypeptides influid and tissue samples by known immunogical procedures. They are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) biochemical activities of cGB-PDE, especially thoseactivities involved in signal transduction. Anti-idiotypic antibodiesspecific for anti-cGB-PDE antibody substances are also contemplated.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor cGB-PDE makes possible the isolation by DNA/DNA hybridization ofgenomic DNA sequences encoding cGB-PDE and specifying cGB-PDE expressioncontrol regulatory sequences such as promoters, operators and the like.DNA/DNA hybridization procedures carried out with DNA sequences of theinvention under stringent conditions are likewise expected to allow theisolation of DNAs encoding allelic variants of cGB-PDE, otherstructurally related proteins sharing one or more of the biochemicaland/or immunological properties specific to cGB-PDE, and non-humanspecies proteins homologous to cGB-PDE. Polynucleotides of the inventionwhen suitably labelled are useful in hybridization assays to detect thecapacity of cells to synthesize cGB-PDE. Polynucleotides of theinvention may also be the basis for diagnostic methods useful foridentifying a genetic alteration(s) in the cGB-PDE locus that underliesa disease state or states. Also made available by the invention areanti-sense polynucleotides relevant to regulating expression of cGB-PDEby those cells which ordinarily express the same.

The DNA and amino acid sequence information provided by the presentinvention also makes possible the systematic analysis of the structureand function of cGB-PDE and definition of those molecules with which itwill interact. Agents that modulate cGB-PDE activity may be identifiedby incubating a putative modulator with lysate from eucaryotic cellsexpressing recombinant cGB-PDE and determining the effect of theputative modulator on cGB-PDE phosphodiesterase activity. In a preferredembodiment the eucaryotic cell lacks endogenous cyclic nucleotidephosphodiesterase activity. Specifically illustrating such a eucaryoticcell is the yeast strain YKS45 which was deposited with the ATCC on May19, 1993 as Accession No. 74225. The selectivity of a compound thatmodulates the activity of the cGB-PDE can be evaluated by comparing itsactivity on the cGB-PDE to its activity on other PDE isozymes. Thecombination of the recombinant cGB-PDE products of the invention withother recombinant PDE products in a series of independent assaysprovides a system for developing selective modulators of cGB-PDE.

Selective modulators may include, for example, antibodies and otherproteins or peptides which specifically bind to the cGB-PDE or cGB-PDEnucleic acid, oligonucleotides which specifically bind to the cGB-PDE orcGB-PDE nucleic acid and other non-peptide compounds (e.g., isloated orsynthetic organic molecules) which specifically react with cGB-PDE orcGB-PDE nucleic acid. Mutant forms of cGB-PDE which affect the enzymaticactivity or cellular localization of the wild-type cGB-PDE are alsocontemplated by the invention. Presently preferred targets for thedevelopment of selective modulators include, for example: (1) theregions of the cGB-PDE which contact other proteins and/or localize thecGB-PDE within a cell, (2) the regions of the cGB-PDE which bindsubstrate, (3) the allosteric cGMP-binding site(s) of cGB-PDE, (4) thephosphorylation site(s) of cGB-PDE and (5) the regions of the cGB-PDEwhich are involved in dimerization of cGB-PDE subunits. Modulators ofcGB-PDE activity may be therapeutically useful in treatment of a widerange of diseases and physiological conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

Numerous other aspects and advantages of the present invention will beapparent upon consideration of the following detailed descriptionthereof, reference being made to the drawing wherein:

FIG. 1A to 1C is an alignment of the conserved catalytic domains ofseveral PDE isoenzymes wherein residues which are identical in all PDEslisted are indicated by their one letter amino acid abbreviation in the“conserved” line, residues which are identical in the cGB-PDE andphotoreceptor PDEs only are indicated by a star in the “conserved” lineand gaps introduced for optimum alignment are indicated by periods;

FIG. 2A to 2C is an alignment of the cGMP-binding domains of several PDEisoenzymes wherein residues which are identical in all PDEs listed areindicated by their one letter amino acid abbreviation in the “conserved”line and gaps introduced for optimum alignment are indicated by periods;

FIG. 3 is an alignment of internally homologous repeats from several PDEisoenzymes wherein residues identical in each repeat A and B from allcGMP-binding PDEs listed are indicated by their one letter amino acidabbreviation in the “conserved” line and stars in the “conserved” linerepresent positions in which all residues are chemically conserved;

FIG. 4 schematically depicts the domain organization of cGB-PDE;

FIG. 5 is a bar graph representing the results of experiments in whichextracts of COS cells transfected with bovine cGB-PDE sequences orextracts of untransfected COS cells were assayed for phosphodiesteraseactivity using either 20 μM cGMP or 20 μM cAMP as the substrate;

FIG. 6 is a graph depicting results of assays of extracts from cellstransfected with bovine cGB-PDE sequences for cGMP phosphodiesteraseactivity in the presence of a series of concentrations ofphosphodiesterase inhibitors including dypyridamole (closed squares),zaprinast (closed circles), methoxymethylxanthine (closed triangles) androlipram (open circles);

FIG. 7 is a bar graph presenting results of experiments in which cellextracts from COS cells transfected with bovine cGB-PDE sequences orcontrol untransfected COS cells were assayed for [³H]cGMP-bindingactivity in the absence (−) or presence (+) of 0.2 mM IBMX; and

FIG. 8 is a graph of the results of assays in which extracts from cellstransfected with bovine cGB-PDE sequences were assayed for[³H]cGMP-binding activity in the presence of excess unlabelled cAMP(open circles) or cGMP (closed circles) at the concentrations indicated.

DETAILED DESCRIPTION

The following examples illustrate the invention. Example 1 describes theisolation of a bovine cGB-PDE cDNA fragment by PCR and subsequentisolation of a full length cGB-PDE cDNA using the PCR fragment as aprobe. Example 2 presents an analysis of the relationship of the bovinecGB-PDE amino acid sequence to sequences reported for various otherPDEs. Northern blot analysis of cGB-PDE mRNA in various bovine tissuesis presented in Example 3. Expression of the bovine cGB-PDE cDNA in COScells is described in Example 4. Example 5 presents results of assays ofthe cGB-PDE COS cell expression product for phosphodiesterase activity,cGMP-binding activity and Zn²⁺ hydrolase activity. Example 6 describesthe isolation of human cDNAs homologous to the bovine cGB-PDE cDNA. Theexpression of a human cGB-PDE cDNA in yeast cells is presented inExample 7. RNase protection assays to detect cGB-PDE in human tissuesare described in Example 8. Example 9 describes the bacterial expressionof human cGB-PDE cDNA and the development of antibodies reactive withthe bacterial cGB-PDE expression product. Example 10 describes cGB-PDEanalogs and fragments. The generation of monoclonal antibodies thatrecognize cGB-PDE is described in Example 11. Example 12 relates toutlilizing recombinant cGB-PDE products of the invention to developagents that selectively modulate the biological activities of cGB-PDE.

EXAMPLE 1

The polymerase chain reaction (PCR) was utilized to isolate a cDNAfragment encoding a portion of cGB-PDE from bovine lung first strandcDNA. Fully degenerate sense and antisense PCR primers were designedbased on the partial cGB-PDE amino acid sequence described in Thomas I,supra, and novel partial amino acid sequence information.

A. Purification of cGB-PDE Protein

cGB-PDE was purified as described in Thomas I, supra, or by amodification of that method as described below.

Fresh bovine lungs (5-10 kg) were obtained from a slaughterhouse andimmediately placed on ice. The tissue was ground and combined with coldPEM buffer (20 mM sodium phosphate, pH 6.8, containing 2 mM EDTA and 25mM β-mercaptoethanol). After homogenization and centrifugation, theresulting supernatant was incubated with 4-7 liters of DEAE-cellulose(Whatman, UK) for 3-4 hours. The DEAE slurry was then filtered undervacuum and rinsed with multiple volumes of cold PEM. The resin waspoured into a glass column and washed with three to four volumes of PEM.The protein was eluted with 100 mM NaCl in PEM and twelve 1-literfractions were collected. Fractions were assayed for IBMX-stimulatedcGMP binding and cGMP phosphodiesterase activities by standardprocedures described in Thomas et al., supra. Appropriate fractions werepooled, diluted 2-fold with cold, deionized water and subjected to BlueSepharose® CL-6B (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.)chromatography. Zinc chelate affinity adsorbent chromatography was thenperformed using either an agarose or Sepharose-based gel matrix. Theresulting protein pool from the zinc chelation step treated as describedin the Thomas I, supra, or was subjected to a modified purificationprocedure.

As decribed in Thomas I, supra, the protein pool was applied in multipleloads to an HPLC Bio-Sil TSK-545 DEAE column (150×21.5 mm) (BioRadLaboratories, Hercules, Calif.) equilibrated in PEM at 4° C. After anequilibration period, a 120-ml wash of 50 mM NaCl in PEM was followed bya 120-ml linear gradient (50-200 mM NaCl in PEM) elution at a flow rateof 2 ml/minute. Appropriate fractions were pooled and concentrated indialysis tubing against Sephadex G-200 (Boehringer MannheimBiochemicals, UK) to a final volume of 1.5 ml. The concentrated cGB-PDEpool was applied to an HPLC gel filtration column (Bio-Sil TSK-250,500×21.5 mm) equilibrated in 100 mM sodium phosphate, pH 6.8, 2 mM EDTA,25 mM β-mercaptoethanol and eluted with a flow rate of 2 ml/minute at 4°C.

If the modified, less cumbersome procedure was performed, the proteinpool was dialyzed against PEM for 2 hours and loaded onto a 10 mlpreparative DEAE Sephacel column (Pharmacia) equilibrated in PEM buffer.The protein was eluted batchwise with 0.5M NaCl in PEM, resulting in anapproximately 10-15 fold concentration of protein. The concentratedprotein sample was loaded onto an 800 ml (2.5 cm×154 cm) Sephacryl S400gel filtration column (Boehringer) equilibrated in 0.1M NaCl in PEM, andeluted at a flow rate of 1.7 ml/minute.

The purity of the protein was assessed by Coomassie staining aftersodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Approximately 0.5-3.0 mg of pure cGB-PDE were obtained per 10 kg bovinelung.

Rabbit polyclonal antibodies specific for the purified bovine cGB-PDEwere generated by standard procedures.

B. Amino Acid Sequencing of cGB-PDE

cGB-PDE phosphorylated with [³²P]ATP and was then digested with proteaseto yield ³²P-labelled phosphopeptides. Approximately 100 μg of purifiedcGB-PDE was phosphorylated in a reaction mixture containing 9 mM MgCl₂,9 μM [³²P]ATP, 10 μM cGMP, and 4.2 μg purified bovine catalytic subunitof cAMP-dependent protein kinase (cAK) in a final volume of 900 μl.Catalytic subunit of cAK was prepared according to the method ofFlockhart et al., pp. 209-215 in Marangos et al., Brain ReceptorMethodologies, Part A, Academic Press, Orlando, Fla. (1984). Thereaction was incubated for 30 minutes at 30° C., and stopped by additionof 60 μl of 200 mM EDTA.

To obtain a first peptide sequence from cGB-PDE, 3.7 μl of a 1 mg/mlsolution of a α-chymotrypsin in KPE buffer (10 mM potassium phosphate,pH 6.8, with 2 mM EDTA) was added to 100 μg purified, phosphorylatedcGB-PDE and the mixture was incubated for 30 minutes at 30° C.Proteolysis was stopped by addition of 50 μl of 10% SDS and 25 μl ofβ-mercaptoethanol. The sample was boiled until the volume was reduced toless than 400 μl, and was loaded onto an 8% preparativeSDS-polyacrylamide gel and subjected to electrophoresis at 50 mAmps. Theseparated digestion products were electroblotted onto Immobilonpolyvinylidene difluoride (Millipore, Bedford, Mass.), according to themethod of Matsudaira, J. Biol. Chem, 262: 10035-10038 (1987).Transferred protein was identified by Coomassie Blue staining, and a 50kDa band was excised from the membrane for automated gas-phase aminoacid sequencing. The sequence of the peptide obtained by theα-chymotryptic digestion procedure is set out below as SEQ ID NO: 1.REXDANRINYMYAQYVKNTM SEQ ID NO: 1

A second sequence was obtained from a cGB-PDE peptide fragment generatedby V8 proteolysis. Approximately 200 μg of purified cGB-PDE was added to10 mM MgCl₂, 10M [³²P]ATP, 100 μM cGMP, and 1 μg/ml purified catalyticsubunit of cAK in a final volume of 1.4 ml. The reaction was incubatedfor 30 minutes at 30° C., and was terminated by the addition of 160 μlof 0.2M EDTA. Next, 9 μl of 1 mg/ml Staphylococcal aureus V8 protease(International Chemical Nuclear Biomedicals, Costa Mesa, Calif.) dilutedin KPE was added, followed by a 15 minute incubation at 30° C.Proteolysis was stopped by addition of 88 μl of 10% SDS and 45 μlβ-mercaptoethanol. The digestion products were separated byelectrophoresis on a preparative 10% SDS-polyacrylamide gel run at 25mAmps for 4.5 hours. Proteins were electroblotted and stained asdescribed above. A 28 kDa protein band was excised from the membrane andsubjected to automated gas-phase amino acid sequencing. The sequenceobtained is set out below as SEQ ID NO: 2. QSLAAAVVP SEQ ID NO: 2C. PCR Amplification of Bovine cDNA

The partial amino acid sequences utilized to design primers (SEQ ID NO:3, below, and amino acids 9-20 of SEQ ID NO: 1) and the sequences of thecorresponding PCR primers (in IUPAC nomenclature) are set below whereinSEQ ID NO: 3 is the sequence reported in Thomas I, supra. SEQ ID NO: 3   F   D   N   D   E   G   E   Q 5′ TTY GAY AAY GAY GAR GGN GAR CA 3′(SEQ ID NO: 4) 3′ AAR CTR TTR CTR CTY CCN CTY GT 5′ (SEQ ID NO: 5) SEQID NO: 1, Amino acids 9-20   N   Y   M   Y   A   Q   Y   V   K   N   T   M 5′ AAY TAY ATG TAY GCNCAR TAY GT 3′ (SEQ ID NO: 6) 3′ TTR ATR TAC ATR CGN GTY ATR CA 5′ (SEQID NO: 7) 3′ TTR ATR TAC ATR CGN GTY ATR CAN TTY TTR TGN TAC 5′ (SEQ IDNO: 8)The sense and antisense primers, synthesized using an Applied BiosystemsModel 380A DNA Synthesizer (Foster City, Calif.), were used in allpossible combinations to amplify cGB-PDE-specific sequences from bovinelung first strand cDNA as described below.

After ethanol precipitation, pairs of oligonucleotides were combined(SEQ ID NO: 4 or 5 combined with SEQ ID NOs: 6, 7 or 8) at 400 nM eachin a PCR reaction. The reaction was run using 50 ng first strand bovinelung cDNA (generated using AMV reverse transcriptase and random primerson oligo dT selected bovine lung mRNA), 200 μM dNTPs, and 2 units of Taqpolymerase. The initial denaturation step was carried out at 94° C. for5 minutes, followed by 30 cycles of a 1 minute denaturation step at 94°C., a two minute annealing step at 50° C., and a 2 minute extension stepat 72° C. PCR was performed using a Hybaid Thermal Reactor (ENKScientific Products, Saratoga, Calif.) and products were separated bygel electrophoresis on a 1% low melting point agarose gel run in 40 mMTris-acetate, 2 mM EDTA. A weak band of about 800-840 bp was seen withthe primers set out in SEQ ID NOs: 4 and 7 and with primers set out inSEQ ID NOs: 4 and 8. None of the other primer pairs yielded visiblebands. The PCR product generated by amplification with the primers setout in SEQ ID NOs: 4 and 7 was isolated using the Gene Clean® (Bio101,La Jolla, Calif.) DNA purification kit according to the manufacturer'sprotocol. The PCR product (20 ng) was ligated into 200 ng of linearizedpBluescript KS(+) (Stratagene, La Jolla, Calif.), and the resultingplasmid construct was used to transform E. coli XL1 Blue cells(Stratagene Cloning Systems, La Jolla, Calif.). Putative transformationpositives were screened by sequencing. The sequences obtained were nothomologous to any known PDE sequence or to the known partial cGB-PDEsequences.

PCR was performed again on bovine lung first strand cDNA using theprimers set out in SEQ ID NOs: 4 and 7. A clone containing a 0.8 Kbinsert with a single large open reading frame was identified. The openreading frame encoded a polypeptide that included the amino acids KNTM(amino acids 17-20 of SEQ ID NO: 1 which were not utilized to design theprimer sequence which is set out in SEQ ID NO: 7) and that possessed ahigh degree of homology to the deduced amino acid sequences of the cGs-,ROS- and COS-PDEs. The clone identified corresponds to nucleotides489-1312 of SEQ ID NO: 9.

D. Construction and Hybridization Screening of a Bovine cDNA Library

In order to obtain a cDNA encoding a full-length cGB-PDE, a bovine lungcDNA library was screened using the ³²P-labelled PCR-generated cDNAinsert as a probe.

Polyadenylated RNA was prepared from bovine lung as described Sonnenburget al., J. Biol. Chem., 266: 17655-17661 (1991). First strand cDNA wassynthesized using AMV reverse transcriptase (Life Sciences, St.Petersburg, Fla.) with random hexanucleotide primers as described inAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York (1987). Second strand cDNA was synthesized using E. coliDNA polymerase I in the presence of E. coli DNA ligase and E. coli RNAseH. Selection of cDNAs larger than 500 bp was performed by Sepharose®CL-4B (Millipore) chromatography. EcoRI adaptors (Promega, Madison,Wis.) were ligated to the cDNA using T4 DNA ligase. Following heatinactivation of the ligase, the cDNA was phosphorylated using T4polynucleotide kinase. Unligated adaptors were removed by Sepharose®CL-4B chromatography (Pharmacia, Piscataway, N.J.). The cDNA was ligatedinto EcoRI-digested, dephosphorylated lambda Zap®II arms (Stratagene)and packaged with Gigapac® Gold (Stratagene) extracts according to themanufacturer's protocol. The titer of the unamplified library was9.9×10⁵ with 18% nonrecombinants. The library was amplified by plating50,000 plaque forming units (pfu) on to twenty 150 mm plates, resultingin a final titer of 5.95×10⁶ pfu/ml with 21% nonrecombinants.

The library was plated on twenty-four 150 mm plates at 50,000 pfu/plate,and screened with the ³²P-labelled cDNA clone. The probe was preparedusing the method of Feinberg et al., Anal. Biochem., 137: 266-267(1984), and the ³²P-labelled DNA was purified using Elutip-D® columns(Schleicher and Schuell Inc., Keene, N.H.) using the manufacturer'sprotocol. Plaque-lifts were performed using 15 cm nitrocellulosefilters. Following denaturation and neutralization, DNA was fixed ontothe filters by baking at 80° C. for 2 hours. Hybridization was carriedout at 42° C. overnight in a solution containing 50% formamide, 5×SSC(0.75M NaCl, 0.75M sodium citrate, pH 7), 25 mM sodium phosphate (pH7.0), 2× Denhardt's solution, 10% dextran sulfate, 90 μg/ml yeast tRNA,and approximately 10⁶ cpm/ml ³²P-labelled probe (5×10⁸ cpm/μg). Thefilters were washed twice in 0.1×SSC, 0.1% SDS at room temperature for15 minutes per wash, followed by a single 20 minute wash in 0.1×SSC, 1%SDS at 45° C. The filters were then exposed to X-ray film at −70° C. forseveral days.

Plaques that hybridized with the labelled probe were purified by severalrounds of replating and rescreening. Insert cDNAs were subcloned intothe pBluescript SK(−) vector (Stratagene) by the in vivo excision methoddescribed by the manufacturer's protocol. Southern blots were performedin order to verify that the rescued cDNA hybridized to the PCR probe.Putative cGB-PDE cDNAs were sequenced using Sequenase® Version 2.0(United States Biochemical Corporation, Cleveland, Ohio) or TaqTrack®kits (Promega).

Three distinct cDNA clones designated cGB-2, cGB-8 and cGB-10 wereisolated. The DNA and deduced amino acid sequences of clone cGB-8 areset out in SEQ ID NOs: 9 and 10. The DNA sequence downstream ofnucleotide 2686 may represent a cloning artifact. The DNA sequence ofcGB-10 is identical to the sequence of cGB-8 with the exception of onenucleotide. The DNA sequence of clone cGB-2 diverges from that of clonecGB-8 5′ to nucleotide 219 of clone cgb-8 (see SEQ ID NO: 9) and couldencode a protein with a different amino terminus.

The cGB-8 cDNA clone is 4474 bp in length and contains a large openreading frame of 2625 bp. The triplet ATG at position 99-101 in thenucleotide sequence is predicted to be the translation initiation siteof the cGB-PDE gene because it is preceded by an in-frame stop codon andthe surrounding bases are compatible with the Kozak consensus initiationsite for eucaryotic mRNAs. The stop codon TAG is located at positions2724-2726, and is followed by 1748 bp of 3′ untranslated sequence. Thesequence of cGB-8 does not contain a transcription termination consensussequence, therefore the clone may not represent the entire 3′untranslated region of the corresponding mRNA.

The open reading frame of the cGB-8 cDNA encodes an 875 amino acidpolypeptide with a calculated molecular mass of 99.5 kD. This calculatedmolecular mass is only slightly larger than the reported molecular massof purified cGB-PDE, estimated by SDS-PAGE analysis to be approximately93 kDa. The deduced amino acid sequence of cGB-8 corresponded exactly toall peptide sequences obtained from purified bovine lung cGB-PDEproviding strong evidence that cGB-8 encodes cGB-PDE.

EXAMPLE 2

A search of the SWISS-PROT and GEnEmb1 data banks (Release of February,1992) conducted using the FASTA program supplied with the GeneticsComputer Group (GCG) Software Package (Madison, Wis.) revealed that onlyDNA and amino acid sequences reported for other PDEs displayedsignificant similarity to the DNA and deduced amino acid of clone cGB-8.

Pairwise comparisons of the cGB-PDE deduced amino acid sequence with thesequences of eight other PDEs were conducted using the ALIGN [Dayhoff etal., Methods Enzymol., 92: 524-545 (1983)] and BESTFIT [Wilbur et al.,Proc. Natl. Acad. Sci. USA, 80: 726-730 (1983)] programs. Like allmammalian phosphodiesterases sequenced to date, cGB-PDE contains aconserved catalytic domain sequence of approximately 250 amino acids inthe carboxyl-terminal half of the protein that is thought to beessential for catalytic activity. This segment comprises amino acids578-812 of SEQ ID NO: 9 and exhibits sequence conservation with thecorresponding regions of other PDEs. Table 1 below sets out the specificidentity values obtained in pairwise comparisons of other PDEs withamino acids 578-812 of cGB-PDE, wherein “ratdunce”is the ratcAMP-specific PDE; “61 kCaM” is the bovine 61 kDacalcium/calmodulin-dependent PDE; “63 kCaM” is the bovine 63 kDacalcium/calmodulin-dependent PDE; “drosdunce” is the drosophilacAMP-specific dunce PDE; “ROS-α” is the bovine ROS-PDE α-subunit;“ROS-β” is the bovine ROS-PDE β-subunit; “COS-α′” is the bovine COS-PDEα′ subunit; and “cGs” is the bovine cGs-PDE (612-844). TABLE 1Phosphodiesterase Catalytic Domain Residues % Identity Ratdunce  77-31631 61 kCaM 193-422 29 63 kcam 195-424 29 drosdunce  1-239 28 ROS-α535-778 45 ROS-β 533-776 46 COS-α′ 533-776 48 cGs 612-844 40

Multiple sequence alignments were performed using the ProgressiveAlignment Algorithm [Feng et al., Methods Enzymol., 183: 375-387 (1990)]implemented in the PILEUP program (GCG Software). FIG. 1A to 1C shows amultiple sequence alignment of the proposed catalytic domain of cGB-PDEwith the all the corresponding regions of the PDEs of Table 1.Twenty-eight residues (see residues indicated by one letter amino acidabbreviations in the “conserved” line on FIGS. 1A to 1C) are invariantamong the isoenzymes including several conserved histidine residuespredicted to play a functional role in catalysis. See Charbonneau etal., Proc. Natl. Acad. Sci. USA, supra. The catalytic domain of cGB-PDEmore closely resembles the catalytic domains of the ROS-PDEs andCOS-PDEs than the corresponding regions of other PDE isoenzymes. Thereare several conserved regions among the photoreceptor PDEs and cGB-PDEthat are not shared by other PDEs. Amino acid positions in these regionsthat are invariant in the photoreceptor PDE and cGB-PDE sequences areindicated by stars in the “conserved” line of FIG. 1A to 1C. Regions ofhomology among cGB-PDE and the ROS- and COS-PDEs may serve importantroles in conferring specificity for cGMP hydrolysis relative to cAMPhydrolysis or for sensitivity to specific pharmacological agents.

Sequence similarity between cGB-PDE, cGs-PDE and the photoreceptor PDEs,is not limited to the conserved catalytic domain but also includes thenoncatalytic cGMP binding domain in the amino-terminal half of theprotein. Optimization of the alignment between cGB-PDE, cGs-PDE and thephotoreceptor PDEs indicates that an amino-terminal conserved segmentmay exist including amino acids 142-526 of SEQ ID NO: 9. Pairwiseanalysis of the sequence of the proposed cGMP-binding domain of cGB-PDEwith the corresponding regions of the photoreceptor PDEs and cGs-PDErevealed 26-28% sequence identity. Multiple sequence alignment of theproposed cGMP-binding domains with the cGMP-binding PDEs is shown inFIG. 2A to 2C wherein abbreviations are the same as indicated forTable 1. Thirty-eight positions in this non-catalytic domain appear tobe invariant among all cGMP-binding PDEs (see positions indicated by oneletter amino acid abbreviations in the “conserved” line of FIG. 2A to2C).

The cGMP-binding domain of the cGMP-binding PDEs contains internallyhomologous repeats which may form two similar but distinct inter- orintra-subunit cGMP-binding sites. FIG. 3 shows a multiple sequencealignment of the repeats a (corresponding to amino acids 228-311 ofcGB-PDE) and b (corresponding to amino acids 410-500 of cGB-PDE) of thecGMP-binding PDEs. Seven residues are invariant in each A and B regions(see residues indicated by one letter amino acid abbreviations in the“conserved” line of FIG. 3). Residues that are chemically conserved inthe A and B regions are indicated by stars in the “conserved” line ofFIG. 3. cGMP analog studies of cGB-PDE support the existence of ahydrogen bond between the cyclic nucleotide binding site on cGB-PDE andthe 2′OH of cGMP.

Three regions of cGB-PDE have no significant sequence similarity toother PDE isoenzymes. These regions include the sequence flanking thecarboxyl-terminal end of the catalytic domain (amino acids 812-875), thesequence separating the cGMP-binding and catalytic domains (amino acids527-577) and the amino-terminal sequence spanning amino acids 1-141. Thesite (the serine at position 92 of SEQ ID NO: 10) of phosphorylation ofcGB-PDE by cGK is located in this amino-terminal region of sequence.Binding of cGMP to the allosteric site on cGB-PDE is required for itsphosphorylation.

A proposed domain structure of cGB-PDE based on the foregoingcomparisons with other PDE isoenzymes is presented in FIG. 4. Thisdomain structure is supported by the biochemical studies of cGB-PDEpurified from bovine lung.

EXAMPLE 3

The presence of cGB-PDE mRNA in various bovine tissues was examined byNorthern blot hybridization.

Polyadenylated RNA was purified from total RNA preparations using thePoly(A) Quick® mRNA purification kit (Stratagene) according to themanufacturer's protocol. RNA samples (5 μg) were loaded onto a 1.2%agarose, 6.7% formaldehyde gel. Electrophoresis and RNA transfer wereperformed as previously described in Sonnenburg et al., supra.Prehybridization of the RNA blot was carried out for 4 hours at 45° C.in a solution containing 50% formamide, 5×SSC, 25 mM sodium phosphate,pH 7, 2× Denhardt's solution, 10% dextran sulfate, and 0.1 mg/ml yeasttRNA. A random hexanucleotide-primer-labelled probe (5×10⁸ cpm/μg) wasprepared as described in Feinberg et al., supra, using the 4.7 kb cGB-8cDNA clone of Example 2 excised by digestion with AccI and SacII. Theprobe was heat denatured and injected into a blotting bag (6×10⁵ cpm/ml)following prehybridization. The Northern blot was hybridized overnightat 45° C., followed by one 15 minute wash with 2×SSC, 0.1% SDS at roomtemperature, and three 20 minute washes with 0.1×SSC, 0.1% SDS at 45° C.The blot was exposed to X-ray film for 24 hours at −70° C. The size ofthe RNA that hybridized with the cGB-PDE probe was estimated using a0.24-9.5 kb RNA ladder that was stained with ethidium bromide andvisualized with UV light.

The ³²P-labelled cGB-PDE cDNA hybridized to a single 6.8 kb bovine lungRNA species. A mRNA band of the identical size was also detected inpolyadenylated RNA isolated from bovine trachea, aorta, kidney andspleen.

EXAMPLE 4

The cGB-PDE cDNA in clone cGB-8 of Example 2 was expressed in COS-7cells (ATCC CRL1651).

A portion of the cGB-8 cDNA was isolated following digestion with therestriction enzyme XbaI. XbaI cut at a position in the pBluescriptpolylinker sequence located 30 bp upstream of the 5′ end of the cGB-8insert and at position 3359 within the cGB-8 insert. The resulting 3389bp fragment, which contains the entire coding region of cGB-8, was thenligated into the unique XbaI cloning site of the expression vector pCDM8(Invitrogen, San Diego, Calif.). The pCDM8 plasmid is a 4.5 kbeucaryotic expression vector containing a cytomegalovirus promoter andenhancer, an SV40-derived origin of replication, a polyadenylationsignal, a procaryotic origin of replication (derived from pBR322) and aprocaryotic genetic marker (supF). E. coli MC1061/P3 cells (Invitrogen)were transformed with the resulting ligation products, andtransformation positive colonies were screened for proper orientation ofthe cGB-8 insert using PCR and restriction enzyme analysis. Theresulting expression construct containing the cGB-8 insert in the properorientation is referred to as pCDM8-cGB-PDE.

The pCDM8-cGB-PDE DNA was purified from large-scale plasmid preparationsusing Qiagen pack-500 columns (Chatsworth, Calif.) according to themanufacturer's protocol. COS-7 cells were cultured in Dulbecco'smodified Eagle's medium (DMEM) containing 10% fetal bovine serum, 50μg/ml penicillin and 50 μg/ml streptomycin at 37° C. in a humidified 5%CO₂ atmosphere. Approximately 24 hours prior to transfection, confluent100 mm dishes of cells were replated at one-fourth or one-fifth theoriginal density. In a typical transfection experiment, cells werewashed with buffer containing 137 mM NaCl, 2.7 mM KCl, 1.1 mM potassiumphosphate, and 8.1 mM sodium phosphate, pH 7.2 (PBS). Then 4-5 ml ofDMEM containing 10% NuSerum (Collaborative Biomedical Products, Bedford,Mass.) was added to each plate. Transfection with 10 μg pCDM8-GB-PDE DNAor PCDM8 vector DNA mixed with 400 μg DEAE-dextran (Pharmacia) in 60 μlTBS [Tris-buffered saline: 25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 5 mMKCl, 0.6 mM Na₂HPO₄, 0.7 mM CaCl₂, and 0.5 mM MgCl₂] was carried out bydropwise addition of the mixture to each plate. The cells were incubatedat 37° C., 5% CO₂ for 4 hours, and then treated with 10% dimethylsulfoxide in PBS for 1 minute. After 2 minutes, the dimethyl sulfoxidewas removed, the cells were washed with PBS and incubated in completemedium. After 48 hours, cells were suspended in 0.5-1 ml of coldhomogenization buffer [40 mM Tris-HCl (pH 7.5), 15 mM benzamidine, 15 mMβ-mercaptoethanol, 0.7 μg/ml pepstatin A, 0.5 μg/ml leupeptin, and 5 μMEDTA] per plate of cells, and disrupted using a Dounce homogenizer. Theresulting whole-cell extracts were assayed for phosphodiesteraseactivity, cGMP-binding activity, and total protein concentration asdescribed below in Example 5.

EXAMPLE 5

Phosphodiesterase activity in extracts of the transfected COS cells ofExample 4 or in extracts of mock transfected COS cells was measuredusing a modification of the assay procedure described for the cGs-PDE inMartins et al., J. Biol. Chem., 257: 1973-1979 (1982). Cells wereharvested and extracts prepared 48 hours after transfection. Incubationmixtures contained 40 mM MOPS buffer (pH 7), 0.8 mM EDTA, 15 mMmagnesium acetate, 2 mg/ml bovine serum albumin, 20 μM [³H]cGMP or[³H]cAMP (100,000-200,000 cpm/assay) and COS-7 cell extract in a totalvolume of 250 μl. The reaction mixture was incubated for 10 minutes at30° C., and then stopped by boiling. Next, 10 μl of 10 mg/ml Crotalusatrox venom (Sigma) was added followed by a 10 minute incubation at 30°C. Nucleoside products were separated from unreacted nucleotides asdescribed in Martins et al., supra. In all studies, less than 15% of thetotal [³H]cyclic nucleotide was hydrolyzed during the reaction.

The results of the assays are presented in FIG. 5 wherein the resultsshown are averages of three separate transfections. Transfection ofCOS-7 cells with pCDM8-cGB-PDE DNA resulted in the expression ofapproximately 15-fold higher levels of cGMP phosphodiesterase activitythan in mock-transfected cells or in cells transfected with pCDM8 vectoralone. No increase in cAMP phosphodiesterase activity over mock orvector-only transfected cells was detected in extracts from cellstransfected with pCDM8-cGB-PDE DNA. These results confirm that thecGB-PDE bovine cDNA encodes a cGMP-specific phosphodiesterase.

Extracts from the transfected COS cells of Example 4 were also assayedfor cGMP PDE activity in the presence of a series of concentrations ofthe PDE inhibitors zaprinast, dipyridamole (Sigma),isobutyl-1-methyl-8-methoxymethylxanthine (MeOxMeMIX) and rolipram.

The results of the assays are presented in FIG. 6 wherein PDE activityin the absence of inhibitor is taken as 100% and each data pointrepresents the average of two separate determinations. The relativepotencies of PDE inhibitors for inhibition of cGMP hydrolysis by theexpressed cGB-BPDE cDNA protein product were identical to those relativepotencies reported for native cGB-PDE purified from bovine lung (ThomasI, supra). IC₅₀ values calculated from the curves in FIG. 6 are asfollows: zaprinast (closed circles), 2 μM; dipyridamole (closedsquares), 3.5 μM; MeOxMeMIX (closed triangles), 30 μM; and rolipram(open circles), >300 μM. The IC₅₀ value of zaprinast, a relativelyspecific inhibitor of cGMP-specific phosphodiesterases, was at least twoorders of magnitude lower than that reported for inhibition ofphosphodiesterase activity of the cGs-PDE or of the cGMP-inhibitedphosphodiesterase (cGi-PDEs) (Reeves et al., pp. 300-316 in Beavo etal., supra). Dipyrimadole, an effective inhibitor of selected cAMP- andcGMP-specific phosphodiesterases, was also a potent inhibitor of theexpressed cGB-PDE. The relatively selective inhibitor ofcalcium/calmodulin-stimulated phosphodiesterase (CaM-PDEs), MeOxMeMIX,was approximately 10-fold less potent than zaprinast and dipyridamole,in agreement with results using cGB-PDE activity purified from bovinelung. Rolipram, a potent inhibitor of low K_(m) cAMP phosphodisterases,was a poor inhibitor of expressed cGB-PDE cDNA protein product. Theseresults show that the cGB-PDE cDNA encodes a phosphodiesterase thatpossesses catalytic activity characteristic of cGB-PDE isolated frombovine tissue, thus verifying the identity of the cGB-8 cDNA clone as acGB-PDE.

It is of interest to note that although the relative potencies of thePDE inhibitors for inhibition of cGMP hydrolysis were identical for therecombinant and bovine isolate cGB-PDE, the absolute IC₅₀ values for allinhibitors tested were 2-7 fold higher for the recombinant cGB-PDE. Thisdifference could not be attributed to the effects of any factors presentin COS-7 cell extracts on cGMP hydrolytic activity, since cGB-PDEisolated from bovine tissue exhibited identical kinetics of inhibitionas a pure enzyme, or when added back to extracts of mock-transfectedCOS-7 cells. This apparent difference in pharmacological sensitivity maybe due to a subtle difference in the structure of the recombinantcGB-PDE cDNA protein product and bovine lung cGB-PDE, such as adifference in post-translational modification at or near the catalyticsite. Alternatively, this difference may be due to an alteration of thecatalytic activity of bovine lung cGB-PDE over several purificationsteps.

Cell extracts were assayed for [H³]cGMP-binding activity in the absenceor presence of 0.2 mM 3-isobutyl-1-methylaxanthine (IBMX) (Sigma), acompetitive inhibitor of cGMP hydrolysis. The cGMP binding assay,modified from the assay described in Thomas I, supra, was conducted in atotal volume of 80 μl. Sixty μl of cell extract was combined with 20 μlof a binding cocktail such that the final concentration of components ofthe mixture were 1 μM [³H]cGMP, 5 μM cAMP, and 10 μM 8-bromo-cGMP. ThecAMP and 8-bromo-cGMP were added to block [³H]cGMP binding to cAK andcGK, respectively. Assays were carried out in the absence and presenceof 0.2 mM IBMX. The reaction was initiated by the addition of the cellextract, and was incubated for 60 minutes at 0° C. Filtration of thereaction mixtures was carried out as described in Thomas I, supra.Blanks were determined by parallel incubations with homogenizationbuffer replacing cell extracts, or with a 100-fold excess of unlabelledcGMP. Similar results were obtained with both methods. Total proteinconcentration of the cell extracts was determined by the method ofBradford, Anal. Biochem., 72:248-254 (1976) using bovine serum albuminas the standard.

Results of the assay are set out in FIG. 7. When measured at 1M [³H]cGMPin the presence of 0.2 mM IBMX, extracts from COS-7 cells transfectedwith pCDM8-cGB-PDE exhibited 8-fold higher cGMP-binding activity thanextracts from mock-transfected cells. No IBMX stimulation of backgroundcGMP binding was observed suggesting that little or no endogenouscGB-PDE was present in the COS-7 cell extracts. In extracts ofpCDM8-cGB-PDE transfected cells cGMP-specific activity was stimulatedapproximately 1.8-fold by the addition of 0.2 mM IBMX. The ability ofIBMX to stimulate cGMP binding 2-5 fold is a distinctive property of thecGMP-binding phosphodisterases.

Cell extracts were assayed as described above for [³H]cGMP-bindingactivity (wherein concentration of [³H]cGMP was 2.5 μM) in the presenceof excess unlabelled cAMP or cGMP. Results are presented in FIG. 8wherein cGMP binding in the absence of unlabelled competitor was takenas 100% and each data point represents the average of three separatedeterminations. The binding activity of the protein product encoded bythe cGB-PDE cDNA was specific for cGMP relative to cAMP. Less than10-fold higher concentrations of unlabelled cGMP were required toinhibit [³H]cGMP binding activity by 50% whereas approximately 100-foldhigher concentrations of cAMP were required for the same degree ofinhibition.

The results presented in this example show that the cGB-PDE cDNA encodesa phosphodiesterase which possesses biochemical activitiescharacteristic of native cGB-PDE.

The catalytic domains of mammalian PDEs and a Drosophila PDE contain twotandem conserved sequences (HX₃HX₂₄-₂₆) that are typical Zn²⁺-bindingmotifs in Zn²⁺ hydrolases such as thermolysin [Vallee and Auld,Biochem., 29: 5647-5659 (1990)]. cGB-PDE binds Zn²⁺ in the presence oflarge excesses of Mg²⁺, Mn²⁺, Fe²⁺, Fe³⁺, Ca²⁺ or Cd²⁺. In the absenceof added metal, cGB-PDE has a PDE activity that is approximately 20% ofthe maximum activity that occurs in the presence of 40 mM Mg²⁺, and thisbasal activity is inhibited by 1,10-phenanthroline or EDTA. Thissuggests that a trace metal(s) accounts for the basal PDE activitydespite exhaustive treatments to remove metals. PDE activity isstimulated by addition of Zn²⁺ (0.02-1 μM) or Co²⁺ (1-20 μM), but not byFe²⁺, Fe²⁺, Ca²⁺, Cd²⁺, or Cu²⁺. Zn²⁺ increases the basal PDE activityup to 70% of the maximum stimulation produced by 40 mM Mg²⁺. Thestimulatory effect of Zn²⁺ in these assays may be compromised by aninhibitory effect that is caused by Zn²⁺ concentrations>1 μM. TheZn²⁺-supported PDE activity and Zn²⁺ binding by cGB-PDE occur at similarconcentrations of Zn²⁺. cGB-PDE thus appears to be a Zn²⁺ hydrolase andZn²⁺ appears to play a critical role in the activity of the enzyme. See,Colbran et al., The FASEB J., 8: Abstract 2148 (Mar. 15, 1994).

EXAMPLE 6

Several human cDNA clones, homologous to the bovine cDNA clone encodingcGB-PDE, were isolated by hybridization under stringent conditions usinga nucleic acid probe corresponding to a portion of the bovine cGB-8clone (nucleotides 489-1312 of SEQ ID NO: 9).

Isolation of cDNA Fragments Encoding Human cGB-PDE

Three human cDNA libraries (two glioblastoma and one lung) in the vectorlambda Zap were probed with the bovine cGB-PDE sequence. ThePCR-generated clone corresponding to nucleotides 484-1312 of SEQ ID NO:9 which is described in Example 1 was digested with EcoRI and SalI andthe resulting 0.8 kb cDNA insert was isolated and purified by agarosegel electrophoresis. The fragment was labelled with radioactivenucleotides using a random primed DNA labelling kit (Boehringer).

The cDNA libraries were plated on 150 mm petri plates at a density ofapproximately 50,000 plaques per plate. Duplicate nitrocellulose filterreplicas were prepared. The prehybridization buffer was 3×SSC, 0.1%sarkosyl, 10× Denhardt's, 20 mM sodium phosphate (pH 6.8) and 50 μg/mlsalmon testes DNA. Prehybridization was carried out at 65° C. for aminimum of 30 minutes. Hybridization was carried out at 65° C. overnightin buffer of the same composition with the addition of 1-5×15 cpm/ml ofprobe. The filters were washed at 65° C. in 2×SSC, 0.1% SDS. Hybridizingplaques were detected by autoradiography. The number of cDNAs thathybridized to the bovine probe and the number of cDNAs screened areindicated in Table 2 below. TABLE 2 cDNA Library Type Positive PlaquesPlaques Screened Human SW 1088 dT-primed 1 1.5 × 10⁶ glioblastoma Humanlung dT-primed 2 1.5 × 10⁶ Human SW 1088 dT-primed 4 1.5 × 10⁶glioblastomaPlasmids designated cgbS2.1, cgbS3.1, cgbL23.1, cgbL27.1 and cgbS27.1were excised in vivo from the lambda Zap clones and sequenced.

Clone cgbS3.1 contains 2060 bp of a PDE open reading frame followed by aputative intron. Analysis of clone cgbS2.1 reveals that it correspondsto clone cgbS3.1 positions 664 to 2060 and extends the PDE open readingframe an additional 585 bp before reading into a putative intron. Thesequences of the putative 5′ untranslated region and the proteinencoding portions of the cgbS2.1 and cgbS3.1 clones are set out in SEQID NOs: 11 and 12, respectively. Combining the two cDNAs yields asequence containing approximately 2.7 kb of an open reading encoding aPDE. The three other cDNAs did not extend any further 5′ or 3′ than cDNAcgbS3.1 or cDNA cgbS2.1.

To isolate additional cDNAs, probes specific for the 5′ end of clonecgbS3.1 and the 3′ end of clone cgbS2.1 were prepared and used to screena SW1088 glioblastoma cDNA library and a human aorta cDNA library. A 5′probe was derived from clone cgbS3.1 by PCR using the primerscgbS3.1S311 and cgbL23.1A1286 whose sequences are set out in SEQ ID NOs:8 and 9, respectively, and below. (SEQ ID NO: 13) Primer cgbS3.1S311 5′GCCACCAGAGAAATGGTC 3′ (SEQ ID NO: 14) Primer cgbL23.1A1286 5′ACAATGGGTCTAAGAGGC 3′The PCR reaction was carried out in a 50 ul reaction volume containing50 pg cgbS3.1 cDNA, 0.2 mM dNTP, 10 ug/ml each primer, 50 mM KCl, 10 mMTris-HCl pH 8.2, 1.5 mM MgCl₂ and Taq polymerase. After an initial fourminute denaturation at 94° C., 30 cycles of one minute at 94° C., twominutes at 50° C. and four minutes at 72° C. were carried out. Anapproximately 0.2 kb fragment was generated by the PCR reaction whichcorresponded to nucleotides 300-496 of clone cgbS3.1.

A 3′ probe was derived from cDNA cgbS2.1 by PCR using the oligoscgbL23.1S1190 and cgbS2.1A231 whose sequences are set out below. (SEQ IDNO: 15) Primer cgbL23.1S1190 5′ TCAGTGCATGTTTGCTGC 3′ (SEQ ID NO: 16)Primer cgbS2.1A231 5′ TACAAACATGTTCATCAG 3′The PCR reaction as carried out similarly to that described above forgenerating the 5′ probe, and yielded a fragment of approximately 0.8 kbcorresponding to nucleotides 1358-2139 of cDNA cgbS2.1. The 3′ 157nucleotides of the PCR fragment (not shown in SEQ ID NO: 12) are withinthe presumptive intron.

The two PCR fragments were purified and isolated by agarose gelelectrophoresis, and were labelled with radioactive nucleotides byrandom priming. A random-primed SW1088 glioblastoma cDNA library(1.5×10⁶ plaques) was screened with the labelled fragments as describedabove, and 19 hybridizing plaques were isolated. An additional 50hybridizing plaques were isolated from a human aorta cDNA library (dTand random primed, Clontech, Palo Alto, Calif.).

Plasmids were excised in vivo from some of the positive lambda Zapclones and sequenced. A clone designated cgbS53.2, the sequence of whichis set out in SEQ ID NO: 17, contains an approximately 1.1 kb insertwhose sequence overlaps the last 61 bp of cgbS3.1 and extends the openreading frame an additional 135 bp beyond that found in cgbS2.1. Theclone contains a termination codon and approximately 0.3 kB of putative3′ untranslated sequence.

Generation of a Composite cDNA Encoding Human cGB-PDE

Clones cgbS3.1, cgbS2.1 and cgbS53.2 were used as described in thefollowing paragraphs to build a composite cDNA that contained a completehuman cGB-PDE opening reading frame. The composite cDNA is designatedcgbmet156-2 and was inserted in the yeast ADH1 expression vector pBNY6N.

First, a plasmid designated cgb stop-2 was generated that contained the3′ end of the cGB-PDE open reading frame. A portion of the insert of theplasmid was generated by PCR using clone cgbS53.2 as a template. The PCRprimers utilized were cgbS2.1S1700 and cgbstop-2. Primer cgbS2.1S1700 5′TTTGGAAGATCCTCATCA 3′ (SEQ ID NO: 18) Primer cgbstop-2 5′ATGTCTCGAGTCAGTTCCGCTTGGCTG 3′ (SEQ ID NO: 19)The PCR reaction was carried out in 50 ul containing 50 pg template DNA,0.2 mM dNTPs, 20 mM Tris-HCl pH 8.2, 10 mM KCl, 6 mM (NH₄)₂SO₄, 1.5 mMMgCl₂, 0.1% Triton-X-100, 500 ng each primer and 0.5 units of Pfupolymerase (Stratagene). The reaction was heated to 94° C. for 4 minutesand then 30 cycles of 1 minute at 94° C., 2 minutes at 50° C. and fourminutes at 72° C. were performed. The polymerase was added during thefirst cycle at 50° C. The resulting PCR product was phenol/chloroformextracted, chloroform extracted, ethanol precipitated and cut with therestriction enzymes BclI and XhoI. The restriction fragment was purifiedon an agarose gel and eluted.

This fragment was ligated to the cDNA cgbS2.1 that had been grown indam⁻ E. coli, cut with the restriction enzymes BclI and XhoI, andgel-purified using the Promega magic PCR kit. The resulting plasmid wassequenced to verify that cgbstop-2 contains the 3′ portion of thecGB-PDE open reading frame.

Second, a plasmid carrying the 5′ end of the human cGB-PDE open readingframe was generated. Its insert was generated by PCR using clone cgbS3.1as a template. PCR was performed as described above using primerscgbmet156 and cgbS2.1A2150. Primer cgbmet156 (SEQ ID NO: 20) 5′TACAGAATTCTGACCATGGAGCGGGCCGGC 3′ Primer cgbS2.1A2150 (SEQ ID NO: 21) 5′CATTCTAAGCGGATACAG 3′The resulting PCR fragment was phenol/choloform extracted, choloformextracted, ethanol precipitated and purified on a Sepharose CL-6Bcolumn. The fragment was cut with the restriction enzymes EcoRV andEcoRI, run on an agarose gel and purified by spinning through glasswool. Following phenol/chloroform extraction, chloroform extraction andethanol precipitation, the fragment was ligated into EcoRI/EcoRVdigested BluescriptII SK(+) to generate plasmid cgbmet156. The DNAsequence of the insert and junctions was determined. The insert containsa new EcoRI site and an additional 5 nucleotides that together replacethe original 155 nucleotides 5′ of the initiation codon. The insertextends to an EcoRV site beginning 531 nucleotides from the initiationcodon.

The 5′ and 3′ portions of the cGB-PDE open reading frame were thenassembled in vector pBNY6a. The vector pBNY6a was cut with EcoRI andXhoI, isolated from a gel and combined with the agarose gel purifiedEcoRI/EcoRV fragment from cgbmet156 and the agarose gel purifiedEcoRV/XhoI fragment from cgbstop-2. The junctions of the insert weresequenced and the construct was named hcbgmet156-2 6a.

The cGB-PDE insert from hcbgmet156-2 6a was then moved into theexpression vector pBNY6n. Expression of DNA inserted in this vector isdirected from the yeast ADH1 promoter and terminator. The vectorcontains the yeast 2 micron origin of replication, the pUC19 origin ofreplication and an ampicillan resistance gene. Vector pBNY6n was cutwith EcoRI and XhoI and gel-purified. The EcoRI/XhoI insert fromhcgbmet156-2 6a was gel purified using Promega magic PCR columns andligated into the cut pBNY6n. All new junctions in the resultingconstruct, hcgbmet156-2 6n, were sequenced. The DNA and deduced aminoacid sequences of the insert of hcgbmet156-2 6n which encodes acomposite human cGB-PDE is set out in SEQ ID NOs: 22 and 23. The insertextends from the first methionine in clone cgbS3.1 (nucleotide 156) tothe stop codon (nucleotide 2781) in the composite cDNA. Because themethionine is the most 5′ methionine in clone cgbS3.1 and because thereare no stop codons in frame with the methionine and upstream of it, theinsert in pBNY6n may represent a truncated form of the open readingframe.

Variant cDNAs

Four human cGB-PDE cDNAs that are different from the hcgbmet156-2 6ncomposite cDNA have been isolated. One cDNA, cgbL23.1, is missing aninternal region of hcgbmet156-2 6n (nucleotides 997-1000 to 1444-1447).The exact end points of the deletion cannot be determined from the cDNAsequence at those positions. Three of the four variant cDNAs have 5′ endsequences that diverge from the hcgbmet156-2 6n sequence upstream ofnucleotide 151 (cDNAs cgbA7f, cgbA5C, cgbI2). These cDNAs presumablyrepresent alteratively spliced or unspliced mRNAs.

EXAMPLE 7

The composite human cGB-PDE cDNA construct, hcgbmet156-2 6n, wastransformed into the yeast strain YKS45 (ATCC 74225) (MATα his3 trp1 ur3leu3 pde1::HIS3 pde2::TRP1) in which two endogenous PDE genes aredeleted. Transformants complementing the leu⁻ deficiency of the YKS45strain were selected and assayed for cGB-PDE activity. Extracts fromcells bearing the plasmid hcgbmet156-2 6n were determined to displaycyclic GMP-specific phosphodiesterase activity by the assay describedbelow.

One liter of YKS45 cells transformed with the plasmid cgbmet156-2 6n andgrown in SC-leu medium to a density of 1-2×10⁷ cells/ml was harvested bycentrifugation, washed once with deionized water, frozen in dryice/ethanol and stored at −70° C. Cell pellets (1-1.5 ml) were thawed onice in the presence of an equal volume of 25 mM Tris-Cl (pH 8.0)/5 mMEDTA/5 mM EGTA/1 mM o-phenanthroline/0.5 mM AEBSF (Calbiochem)/0.1%β-mercaptoethanol and 10 ug/ml each of aprotinin, leupeptin, andpepstatin A. The thawed cells were added to 2 ml of acid-washed glassbeads (425-600 μM, Sigma) in 15 ml Corex tube. Cells were broken with 4cycles consisting of a 30 second vortexing on setting 1 followed by a 60second incubation on ice. The cell lysate was centrifuged at 12,000×gfor 10 minutes and the supernatant was passed through a 0.8μ filter. Thesupernatant was assayed for cGMP PDE activity as follows. Samples wereincubated for 20 minutes at 30° C. in the presence of 45 mM Tris-Cl (pH8.0), 2 mM EGTA, 1 mM EDTA, 0.2 mg/ml BSA, 5 mM MgCl₂, 0.2 mMo-phenanthroline, 2 ug/ml each of pepstatin A, leupeptin, and aprotinin,0.1 mM AEBSF, 0.02% β-mercaptoethanol and 0.1 mM [³H]cGMP as substrate.[¹⁴C]-AMP (0.5 nCi/assay) was added as a recovery standard. The reactionwas terminated with stop buffer (0.1M ethanolamine pH 9.0, 0.5M ammoniumsulfate, 10 mM EDTA, 0.05% SDS final concentration). The product wasseparated from the cyclic nucleotide substrate by chromatography onBioRad Affi-Gel 601. The sample was applied to a column containingapproximately 0.25 ml of Affi-Gel 601 equilibrated in column buffer(0.1M ethanolamine pH 9.0 containing 0.5M ammonium sulfate). The columnwas washed five times with 0.5 ml of column buffer. The product waseluted with four 0.5 ml aliquots of 0.25 acetic acid and mixed with 5 mlEcolume (ICN Biochemicals). The radioactive product was measured byscintillation counting.

EXAMPLE 8

Analysis of expression of cGB-PDE mRNA in human tissues was carried outby RNase protection assay.

A probe corresponding to a portion of the putative cGMP binding domainof cGB-PDE (402 bp corresponding to nucleotides 1450 through 1851 of SEQID NO: 13) was generated by PCR. The PCR fragment was inserted into theEcoRI site of the plasmid pBSII SK(−) to generate the plasmid RP3. RP3plasmid DNA was linearized with XbaI and antisense probes were generatedby a modification of the Stratagene T7 RNA polymerase kit. Twenty-fiveng of linearized plasmid was combined with 20 microcuries of alpha ³²PrUTP (800 Ci/mmol, 10 mCi/ml), 1× transcription buffer (40 mM Tris Cl,pH 8, 8 mM MgCl₂, 2 mM spermidine, 50 mM NaCl), 0.25 mM each rATP, rGTPand rCTP, 0.1 units of RNase Block II, 5 mM DTT, 8 μM rUTP and 5 unitsof T7 RNA Polymerase in a total volume of 5 μl. The reaction was allowedto proceed 1 hour at room temperature and then the DNA template wasremoved by digestion with RNase free DNase. The reaction was dilutedinto 100 μl of 40 mM Tris Cl, pH 8, 6 mM MgCl₂ and 10 mM NaCl. Fiveunits of RNase-free DNase were added and the reaction was allowed tocontinue another 15 minutes at 37° C. The reaction was stopped by aphenol extraction followed by a phenol chloroform extraction. One halfvolume of 7.5M NH₄OAc was added and the probe was ethanol precipitated.

The RNase protection assays were carried out using the Ambion RNaseProtection kit (Austin, Tex.) and 10 μg RNA isolated from human tissuesby an acid guanidinium extraction method. Expression of cGB-PDE mRNA waseasily detected in RNA extracted from skeletal muscle, uterus, bronchus,skin, right saphenous vein, aorta and SW1088 glioblastoma cells. Barelydetectable expression was found in RNA extracted from right atrium,right ventricle, kidney cortex, and kidney medulla. Only completeprotection of the RP3 probe was seen. The lack of partial protectionargues against the cDNA cgbL23.1 (a variant cDNA described in Example 7)representing a major transcript, at least in these RNA samples.

EXAMPLE 9

Polyclonal antisera was raised to E. coli-produced fragments of thehuman cGB-PDE.

A portion of the human cGB-PDE cDNA (nucleotides 1668-2612 of SEQ ID NO:22, amino acids 515-819 of SEQ ID NO: 23) was amplified by PCR andinserted into the E. coli expression vector pGEX2T (Pharmacia) as aBamHI/EcoRI fragment. The pGEX2T plasmid carries an ampicillinresistance gene, an E. coli laq I^(q) gene and a portion of theSchistosoma japonicum glutathione-S-transferase (GST) gene. DNA insertedin the plasmid can be expressed as a fusion protein with GST and canthen be cleaved from the GST portion of the protein with thrombin. Theresulting plasmid, designated cgbPE3, was transformed into E. colistrain LE392 (Stratagene). Transformed cells were grown at 37° C. to anOD600 of 0.6. IPTG (isopropylthioalactopyranoside) was added to 0.1 mMand the cells were grown at 37° C. for an additional 2 hours. The cellswere collected by centrifugation and lysed by sonication. Cell debriswas removed by centrifugation and the supernatant was fractionated bySDS-PAGE. The gel was stained with cold 0.4M KCl and the GST-cgb fusionprotein band was excised and electroeluted. The PDE portion of theprotein was separated from the GST portion by digestion with thrombin.The digest was fractionated by SDS-PAGE, the PDE protein waselectroeluted and injected subcutaneously into a rabbit. The resultantantisera recognizes both the bovine cGB-PDE fragment that was utilizedas antigen and the full length human cGB-PDE protein expressed in yeast(see Example 8).

EXAMPLE 10

Polynucleotides encoding various cGB-PDE analogs and cGB-PDE fragmentswere generated by standard methods.

A. cGB-PDE Analogs

All known cGMP-binding PDEs contain two internally homologous tandemrepeats within their putative cGMP-binding domains. In the bovinecGB-PDE of the invention, the repeats span at least residues 228-311(repeat A) and 410-500 (repeat B) of SEQ ID NO: 10. Site-directedmutagenesis of an aspartic acid that is conserved in repeats A and B ofall known cGMP-binding PDEs was used to create analogs of cGB-PDE havingeither Asp-289 replaced with Ala (D289A) or Asp478 replaced with Ala(D478A). Recombinant wild type (WT) bovine and mutant bovine cGB-PDEswere expressed in COS-7 cells. cGB-PDE purified from bovine lung (nativecGB-PDE) and WT cGB-PDE displayed identical cGMP-binding kinetics with aK_(d) of approximately 2 μM and a curvilinear dissociation profile(t_(1/2)=1.3 hours at 4° C.). This curvilinearity may have been due tothe presence of distinct high affinity (slow) and low affinity (fast)sites of cGMP binding. The D289A mutant had significantly decreasedaffinity for cGMP (K_(c)>20 μM) and a single rate of cGMP-association(t_(1/2)=0.5 hours), that was similar to that calculated for the fastsite of WT and native cGB-PDE. This suggested the loss of a slowcGMP-binding site in repeat A of this mutant. Conversely, the D478Amutant showed higher affinity for cGMP (K_(d) of approximately 0.5 μM)and a single cGMP-dissociation rate (t_(1/2)=2.8 hours) that was similarto the calculated rate of the slow site of WT and native cGB-PDE. Thissuggested the loss of a fast site when repeat B was modified. Theseresults indicate that dimeric cGB-PDE possesses two homologous butkinetically distinct cGMP-binding sites, with the conserved asparticacid being critical for interaction with cGMP at each site. See, Colbranet al., FASEB J., 8: Abstract 2149 (May 15, 1994).

B. Amino-Terminal Truncated cGB-PDE Polypeptides

A truncated human cGB-PDE polypeptide including amino acids 516-875 ofSEQ ID NO: 23 was expressed in yeast. A cDNA insert extending from theNcoI site at nucleotide 1555 of SEQ ID NO: 22 through the XhoI site atthe 3′ end of SEQ ID NO: 22 was inserted into the ADH2 yeast expressionvector YEpC-PADH2d [Price et al., Meth. Enzymol., 185: 308-318 (1990)]that had been digested with NcoI and SalI to generate plasmidYEpC-PADH2d HcGB. The plasmid was transformed into spheroplasts of theyeast strain yBJ2-54 (prc1-407 prb1-1122 pep4-3 leu2 trp1 ura3-52Δpde1::URA3, HIS3 Δpde2::TRP1 cir*). The endogenous PDE genes aredeleted in this strain. Cells were grown in SC-leu media with 2% glucoseto 10⁷ cells/ml, collected by filtration and grown 24 hours in YEP mediacontaining 3% glycerol. Cells were pelleted by centrifugation and storedfrozen. Cells were disrupted with glass beads and the cell homogenatewas assayed for phosphodiesterase activity essentially as described inPrpic et al., Anal. Biochem., 208: 155-160 (1993). The truncated humancGB-PDE polypeptide exhibited phosphodiesterase activity.

C. Carboxy-Terminal Truncated cGB-PDE Polypeptides

Two different plasmids encoding carboxy-terminal truncated human cGB-PDEpolypeptides were constructed.

Plasmid pBJ6-84Hin contains a cDNA encoding amino acids 1-494 of SEQ IDNO: 23 inserted into the NcoI and SalI sites of vector YEpC-PADH2d. ThecDNA insert extends from the NcoI site at nucleotide position 10 of SEQID. NO: 22 through the HindIII site at nucleotide position 1494 of SEQID NO: 22 followed by a linker and the SalI site of YEpC-PADH2d.

Plasmid pBJ6-84Ban contains a cDNA encoding amino acids 1-549 of SEQ IDNO: 23 inserted into the NcoI and SalI sites of vector YEpC-PADH2d. ThecDNA insert extends from the NcoI site at nucleotide position 10 of SEQID NO: 22 through the BanI site at nucleotide position 1657 of SEQ IDNO: 22 followed by a linker and the SalI site of YEpC-PADH2d.

The trucated cGB-PDE polypeptides are useful for screening formodulators of cGB-PDE activity.

EXAMPLE 11

Monoclonal antibodies reactive with human cGB-PDE were generated.

Yeast yBJ2-54 containing the plasmid YEpADH2 HcGB (Example 10B) werefermented in a New Brunswick Scientific 10 liter Microferm. The cGB-PDEcDNA insert in plasmid YEpADH2 HcGB extends from the NcoI site atnucleotide 12 of SEQ ID NO: 22 to the XhoI site at the 3′ end of SEQ IDNO: 22. An inoculum of 4×10⁹ cells was added to 8 liters of mediacontaining SC-leu, 5% glucose, trace metals, and trace vitamins.Fermentation was maintained at 26° C., agitated at 600 rpm with thestandard microbial impeller, and aerated with compressed air at 10volumes per minute. When glucose decreased to 0.3% at 24 hourspost-inoculation the culture was infused with 2 liters of 5×YEP mediacontaining 15% glycerol. At 66 hours post-inoculation the yeast from theferment was harvested by centrifugation at 4,000×g for 30 minutes at 4°C. Total yield of biomass from this fermentation approached 350 g wetweight.

Human cGB-PDE enzyme was purified from the yeast cell pellet. Assays forPDE activity using 1 mM cGMP as substrate was employed to follow thechromatography of the enzyme. All chromatographic manipulations wereperformed at 4° C.

Yeast (29 g wet weight) were resuspended in 70 ml of buffer A (25 mMTris pH 8.0, 0.25 mM DTT, 5 mM MgCl₂, 10 μM ZnSO⁴, 1 mM benzamidine) andlysed by passing through a microfluidizer at 22-24,000 psi. The lysatewas centrifuged at 10,000×g for 30 minutes and the supernatant wasapplied to a 2.6×28 cm column containing Pharmacia Fast Flow Q anionexchange resin equilibrated with buffer B containing 20 mMBisTris-propane pH 6.8, 0.25 mM DTT, 1 mM MgCl₂, and 10 μM ZnSO₄. Thecolumn was washed with 5 column volumes of buffer B containing 0.125MNaCl and then developed with a linear gradient from 0.125 to 1.0M NaCl.Fractions containing the enzyme were pooled and applied directly to a5×20 cm column of ceramic hydroxyapatite (BioRad) equilibrated in bufferC containing 20 mM BisTris-propane pH 6.8, 0.25 mM DTT, 0.25MKCl, 1 mMMgCl₂, and 10 μM ZnSO₄. The column was washed with 5 column volumes ofbuffer C and eluted with a linear gradient from 0 to 250 mM potassiumphosphate in buffer C. The pooled enzyme was concentrated 8-fold byultrafiltration (YM30 membrane, Amicon). The concentrated enzyme waschromatographed on a 2.6×90 cm column of Pharmacia Sephacryl S300(Piscataway, N.J.) equilibrated in 25 mM BisTris-propane pH 6.8, 0.25 mMDTT, 0.25M NaCl, 1 mM MgCl₂, and 20 μM ZnSO₄. Approximately 4 mg ofprotein was obtained. The recombinant human cGB-PDE enzyme accounted forapproximately 90% of protein obtained as judged by SDS polyacrylamidegel electrophoresis followed by Coomassie blue staining.

The purified protein was used as an antigen to raise monoclonalantibodies. Each of 19 week old Balb/c mice (Charles River BiotechnicalServices, Inc., Wilmington, Mass.) was immunized sub-cutaneously with 50ug purified human cGB-PDE enzyme in a 200 ul emulsion consisting of 50%Freund's complete adjuvant (Sigma Chemical Co.). Subsequent boosts onday 20 and day 43 were administered in incomplete Freund's adjuvant. Apre-fusion boost was done on day 86 using 50 ug enzyme in PBS. Thefusion was performed on day 90.

The spleen from mouse #1817 was removed sterilely and placed in 10 mlserum free RPMI 1640. A single-cell suspension was formed and filteredthrough sterile 70-mesh Nitex cell strainer (Becton Dickinson,Parsippany, N.J.), and washed twice by centrifuging at 200 g for 5minutes and resuspending the pellet in 20 ml serum free RPMI. Thymocytestaken from 3 naive Balb/c mice were prepared in a similar manner.

NS-1 myeloma cells, kept in log phase in RPMI with 11% Fetalclone (FBS)(Hyclone Laboratories, Inc., Logan, Utah) for three days prior tofusion, were centrifuged at 200 g for 5 minutes, and the pellet waswashed twice as described in the foregoing paragraph. After washing,each cell suspension was brought to a final volume of 10 ml in serumfree RPMI, and 20 μl was diluted 1:50 in 1 ml serum free RPMI. 20 μl ofeach dilution was removed, mixed with 20 μl 0.4% trypan blue stain in0.85% saline (Gibco), loaded onto a hemocytometer (Baxter HealthcareCorp., Deerfield, Ill.) and counted.

Two×10⁸ spleen cells were combined with 4.0×10⁷ NS-1 cells, centrifugedand the supernatant was aspirated. The cell pellet was dislodged bytapping the tube and 2 ml of 37° C. PEG 1500 (50% in 75 mM Hepes, pH8.0) (Boehringer Mannheim) was added with stirring over the course of 1minute, followed by adding 14 ml of serum free RPMI over 7 minutes. Anadditional 16 ml RPMI was added and the cells were centrifuged at 200 gfor 10 minutes. After discarding the supernatant, the pellet wasresuspended in 200 ml RPMI containing 15% FBS, 100 μM sodiumhypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ thymocytes/ml. Thesuspension was first placed in a T225 flask (Corning, United Kingdom) at37° C. for two hours before being dispensed into ten 96-well flat bottomtissue culture plates (Corning, United Kingdom) at 200 μl/well. Cells inplates were fed on days 3, 4, 5 post fusion day by aspiratingapproximately 100 μl from each well with an 20 G needle (BectonDickinson), and adding 100 μl/well plating medium described above exceptcontaining 10 units/ml IL-6 and lacking thymocytes.

The fusion was screened initially by ELISA. Immulon 4 plates (Dynatech)were coated at 4° C. overnight with purified recombinant human cGB-PDEenzyme (100 ng/well in 50 mM carbonate buffer pH9.6). The plates werewashed 3× with PBS containing 0.05% Tween 20 (PBST). The supernatantsfrom the individual hybridoma wells were added to the enzyme coatedwells (50 μl/well). After incubation at 37° C. for 30 minutes, andwashing as above, 50 μl of horseradish peroxidase conjugated goatanti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted1:3500 in PBST was added. Plates were incubated as above, washed 4× withPBST and 100 μl substrate consisting of 1 mg/ml o-phenylene diamine(Sigma) and 0.1 μl/ml 30% H₂O₂ in 100 mM citrate, pH 4.5, was added. Thecolor reaction was stopped in 5 minutes with the addition of 50 μL¹ of15% H₂SO₄. A₄₉₀ was read on a plate reader (Dynatech).

Wells C5G, E4D, F1G, F9H, F11G, J4A, and J5D were picked and renamed102A, 102B, 102C, 102D, 102E, 102F, and 102G respectively, cloned two orthree times, successively, by doubling dilution in RPMI, 15% FBS, 100 μMsodium hypoxanathine, 16 μM thymidine, and 10 units/ml IL-6. Wells ofclone plates were scored visually after 4 days and the number ofcolonies in the least dense wells were recorded. Selected wells of theeach cloning were tested by ELISA.

The monoclonal antibodies produced by above hybridomas were isotyped inan ELISA assay. Results showed that monoclonal antibodies 102A to 102Ewere IgG1, 102F was IgG2b and 102G was IgG2a.

All seven monoclonal antibodies reacted with human cGS-PDE as determinedby Western analysis.

EXAMPLE 12

Developing modulators of the biological activities of specific PDEsrequires differentiating PDE isozymes present in a particular assaypreparation. The classical enzymological approach of isolating PDEs fromnatural tissue sources and studying each new isozyme is hampered by thelimits of purification techniques and the inability to definitivelyassess whether complete resolution of a isozyme has been achieved.Another approach has been to identify assay conditions which might favorthe contribution of one isozyme and minimize the contribution of othersin a preparation. Still another approach has been the separation of PDEsby immunological means. Each of the foregoing approaches fordifferentiating PDE isozymes is time consuming and technicallydifficult. As a result many attempts to develop selective PDE modulatorshave been performed with preparations containing more than one isozyme.Moreover, PDE preparations from natural tissue sources are susceptibleto limited proteolysis and may contain mixtures of active proteolyticproducts that have different kinetic, regulatory and physiologicalproperties than the full length PDEs.

Recombinant cGB-PDE polypeptide products of the invention greatlyfacilitate the development of new and specific cGB-PDE modulators. Theuse of human recombinant enzymes for screening for modulators has manyinherent advantages. The need for purification of an isozyme can beavoided by expressing it recombinantly in a host cell that lacksendogenous phosphodiesterase activity (e.g., yeast strain YKS45deposited as ATCC 74225). Screening compounds against human proteinavoids complications that often arise from screening against non-humanprotein where a compound optimized on a non-human protein may fail to bespecific for or react with the human protein. For example, a singleamino acid difference between the human and rodent ⁵HT_(1B) serotoninreceptors accounts for the difference in binding of a compound to thereceptors. [See Oskenberg et al. Nature, 360: 161-163 (1992)]. Once acompound that modulates the activity of the cGB-PDE is discovered, itsselectivity can be evaluated by comparing its activity on the cGB-PDE toits activity on other PDE isozymes. Thus, the combination of therecombinant cGB-PDE products of the invention with other recombinant PDEproducts in a series of independent assays provides a system fordeveloping selective modulators of cGB-PDE. Selective modulators mayinclude, for example, antibodies and other proteins or peptides whichspecifically bind to the cGB-PDE or cGB-PDE nucleic acid,oligonucleotides which specifically bind to the cGB-PDE (see PatentCooperation Treaty International Publication No. WO93/05182 publishedMar. 18, 1993 which describes methods for selecting oligonucleotideswhich selectively bind to target biomolecules) or cGB-PDE nucleic acid(e.g., antisense oligonucleotides) and other non-peptide natural orsynthetic compounds which specifically bind to the cGB-PDE or cGB-PDEnucleic acid. Mutant forms of the cGB-PDE which alter the enzymaticactivity of the cGB-PDE or its localization in a cell are alsocontemplated. Crystallization of recombinant cGB-PDE alone and bound toa modulator, analysis of atomic structure by X-ray crystallography, andcomputer modelling of those structures are methods useful for designingand optimizing non-peptide selective modulators. See, for example,Erickson et al., Ann. Rep. Med. Chem., 27: 271-289 (1992) for a generalreview of structure-based drug design.

Targets for the development of selective modulators include, forexample: (1) the regions of the cGB-PDE which contact other proteinsand/or localize the cGB-PDE within a cell, (2) the regions of thecGB-PDE which bind substrate, (3) the allosteric cGMP-binding site(s) ofcGB-PDE, (4) the metal-binding regions of the cGB-PDE, (5) thephosphorylation site(s) of cGB-PDE and (6) the regions of the cGB-PDEwhich are involved in dimerization of cGB-PDE subunits.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the appended claims should be placed on the invention.

1. A purified and isolated polynucleotide encoding cGB-PDE.
 2. Thepolynucleotide of claim 1 which is a DNA sequence.
 3. The DNA sequenceof claim 2 which is a cDNA sequence or a biological replica thereof. 4.The DNA sequence of claim 2 which is a genomic DNA sequence or abiological replica thereof.
 5. An RNA transcript of the genomic DNAsequence of claim
 4. 6. The DNA sequence of claim 2 which is a wholly orpartially chemically synthesized DNA sequence or a biological replicathereof.
 7. The DNA sequence of claim 4 further comprising an endogenousexpression control DNA sequence.
 8. A DNA vector comprising a DNAsequence according to claim
 2. 9. The vector of claim 8 wherein said DNAsequence is operatively linked to an expression control DNA sequence.10. A host cell stably transformed or transfected with a DNA sequenceaccording to claim 7 in a manner allowing the expression in said hostcell of cGB-PDE polypeptide possessing a ligand/receptor bindingbiological activity or immunological property specific to cGB-PDE.
 11. Amethod for producing cGB-PDE polypeptide, said method comprising growinga host cell according to claim 10 in a suitable nutrient medium andisolating cGB-PDE polypeptide from said cell or the medium of itsgrowth.
 12. A polypeptide or peptide capable of specifically binding tocGB-PDE.
 13. An antibody substance according to claim
 12. 14. Amonoclonal antibody according to claim
 13. 15. A hybridoma cell lineproducing a monoclonal antibody according to claim
 14. 16. A humanizedantibody substance according to claim
 13. 17. An antisensepolynucleotide specific for a polynucleotide encoding cGB-PDE.
 18. A DNAsequence encoding cGB-PDE and selected from the group consisting of: (a)the DNA sequence set out in SEQ ID NO: 9 or 22; (b) a DNA whichhybridizes under stringent conditions to the DNA of (a); and (c) a DNAsequence which, but for the redundancy of the genetic code, wouldhybridize under stringent conditions to a DNA sequence of (a) or (b).